Identification and quantification of low levels of zinc ions is important for biological research, diagnosis of metal ion induced disease state, accurate quantification in foodstuff and environmental samples. The zinc(II) ion (Zn2+) plays an important role in biology and nutrition and when present in high concentrations in the environment can have a toxic effect on biological systems (Berg, J M, et al. Science (1996) 271:1081-5).
Zn2+ is known to play an important role in protein structure, gene regulation, protein synthesis, intracellular protein trafficking, hormone function and immune function and is considered an essential element wherein the USDA recommends daily consumption of a small amount of Zn2+ (Kirschke C P, et al., J Biol Chem (2003) 278(6): 4096-4102). Recently Zn2+ has emerged as an important player in neurotransmission (Frederickson, C J. Int Rev Neurobiol (1989) 31: 145-238) and neural injury (Frederickson C J, et al. J Nutr. (2000) 130:1471-83; Weiss J H, et al. Trends Pharm Sci. (2000) 21:395-400). Much of the total biological zinc is tightly bound to proteins and enzymes (Outten C E, et al. Science (2001) 292:2488-92). Rapid rises in intracellular free Zn2+ ([Zn2+]i) have been linked to neuronal injury in transient global ischemia and epilepsy (Frederickson, C J. Int Rev Neurobiol; Weiss J H, et al. Trends Pharm Sci. (supra)). The mechanisms by which Zn2+ exerts potent neurotoxic effects are still largely unknown. It has been suggested that among the intracellular targets of Zn2+ dependent neurotoxicity, Zn2+ sequestration into mitochondria may play a critical role (Manev H, et al. Exp Neurol (1997) 146:171-8; Sensi S L, et al., PNAS (1999) 96:2414-9; Sensi S L, et al. Eur J Neurosci (2000) 12:3813-8). As with Ca2+, upon excessive cytosolic Zn2+ loading, mitochondria take up Zn2+ and help to restore intracellular Zn2+ homeostasis (Sensi S L, et al. Eur J Neurosci, supra). However, once in the mitochondria, Zn2+ can trigger a prolonged disruption of the functioning of these organelles. Indeed, Zn2+ has been shown to have potent effects on mitochondria (Skulachev V P, et al. Biochem Biophys Res Commun (1967) 26:1-6; Kleiner D. Arch Biochem Biophys (1974) 165:121-5; Link T A, et al. J Biol Chem (1995) 270:25001-6; Wudarczyk J, et al. Arch Biochem Biophys (1999) 363:1-8; Brown A M, et al. J Biol Chem (2000) 275:13441-7; Jiang D, et al. J Biol Chem (2001) 276: 47524-9). For instance, in neuronal mitochondria, rises in [Zn2+]m promote loss of mitochondrial membrane potential (ΔΨm) and generation of reactive oxygen species (ROS) as well as release of pro-apoptotic factors (Sensi S L, et al. Neurobiol Dis (2002) 10:100-108).
Due to the low concentration of physiological free Zn2+ (nanomolar), elucidation of the biology of zinc has been hampered by a lack of suitable detection and imaging reagents. Thus, at least for biological research there is need for more sensitive indicators than have previously been available. Indicators that have been previously used include fluorescent sulfonamides of 8-aminoquinolines (Fahrni C J, et al JACS (1999) 121:11448; Snitsarev V. et al., Biophys J. (2001) 80:1538; Budde T, Neuroscience (1997) 79:347; Frederickson C J., et al. (1987) 20:91. These indicators are UV excitable, potentially damaging, and exhibit low signal levels. Alternatively, visible light indicators have been developed that demonstrate a higher affinity for Zn2+ and a brighter signal than the 8-aminoquinolines based indicators (Burdette S C, eta I., JACS (2001) 123(32):7831-41; Hirano T, et al., Angew Chem Int Ed Engl (2000) 39(6):1052-1054; Maruyama S. et al., JACS (2002) 124(36):10650-1; Hirano T, et al., JACS (2002) 124(23):6555-62). However, at least some of these visible light indicators are only useful in a limited pH range and tend to localize in intracellular acidic compartments. This is due to the chelator moieties of the indicator that become significantly protonated at physiological pH.
Therefore, there is a need for zinc-binding compounds that selectively bind zinc ions in the presence of other physiological relevant metal ions, have a high affinity for physiological concentrations of zinc ions (nanomolar concentrations), can be effectively utilized at a physiological pH and are environmentally sensitive to zinc ions, i.e. show a significant change in signal after being bound by zinc ions.
Herein we disclose novel zinc-binding compounds that selectively bind zinc ions in the presence of physiological concentrations of calcium. These zinc-binding compounds provide powerful tools for selectively binding zinc ions in intracellular compartments, biological fluids and environmental samples. These compounds overcome the limitations of known zinc indicators. Specifically, zinc-binding compounds are disclosed that localize to the mitochondrial membrane and selectively bind zinc ions.